practicals

Question 1

how can you tell the difference between a digested and undigested sample on a gel?

Answer 1

undigested DNA is represented by a sharp band, smearing represents digested DNA

Question 2

when should you disconnect the electrode from the power supply when running a gel?

Answer 2

when the dye has moved 2/3 of the way

Question 3

what is the role of ethidium bromide in an electrophoresis gel?

Answer 3

fluorescent tag

Question 4

what is a polyanion?

Answer 4

a molecule or chemical complex which has negative charges at several sites

Question 5

what is the role of EDTA in stop solution?

Answer 5

chelates Mg2+

Question 6

how do you calculate the length in base pairs?

Answer 6

M(Da)/650

Question 7

what is the beer lambert law? (rearranged)

Answer 7

c=A/molar coefficient x L

Question 8

name the techniques that can be used to determine the catalytic activity of an enzyme

Answer 8

spectrophotometry, isotopic labelling, titration, quantitative chemical analysis

Question 9

what are the advantages of spectrophotometric techniques?

Answer 9

reactions can be followed continuously
rapid
low concentrations measurable

Question 10

what does the x-intercept represent on a lineweaver-burk plot?

Answer 10

-1/Km

Question 11

what does the y-intercept represent on a lineweaver-burk plot?

Answer 11

1/Vmax

Question 12

what effect does a non-competitive inhibitor have on Vmax and Km?

Answer 12

Vmax = reduced
Km= same

Question 13

what effect does a competitive inhibitor have on Vmax and Km?

Answer 13

Vmax= same
Km= increased

Question 14

what does Km effectively measure?

Answer 14

affinity of an enzyme for the substrate

Question 15

what is the limitation of lineweaver-burk plots?

Answer 15

they lay undue emphasis on data obtained at low substrate concentrations

Question 16

how is activity of B-Galactosidase measured?

Answer 16

measuring ONP production by hydrolysis of the artificial substrate ONPG

Question 17

why is turkey blood used when purifying histone proteins?

Answer 17

turkey RBCs are nucleate, mammalian RBCs are not

Question 18

what is AEBSF?

Answer 18

a water soluble protease inhibitor

Question 19

what is Triton X-100?

Answer 19

detergent used to lyse cells so histones can be extracted

Question 20

why is micrococcal nuclease digestion carried out when purifying histone proteins?

Answer 20

targets nucleic acids not histones

Question 21

How does SDS allow proteins to be separated by mass?

Answer 21

creates denaturing conditions that separate proteins by molecular weight

Question 22

which technique is used to separate histone proteins?

Answer 22

electrophoresis through a polyacrylamide gel

Question 23

what is the first step when separating histone proteins?

Answer 23

incubation and washing in buffer to break cell membrane and denature proteins not associated with histones

Question 24

what is the role of MNase digestion in the separation of histones?

Answer 24

digests insoluble chromatin into insoluble oligonucleosomes

Question 25

name two factors which affect the rate at which proteins move through a polyacrylamide gel

Answer 25

molecular weight and the presence of charged side chains